Cumulus Extracellular Matrix Is an Important Part of Oocyte Microenvironment in Ovarian Follicles: Its Remodeling and Proteolytic Degradation

Abstract

The extracellular matrix (ECM) is an essential structure with biological activities. It has been shown that the ECM influences gene expression via cytoskeletal components and the gene expression is dependent upon cell interactions with molecules and hormones. The development of ovarian follicles is a hormone dependent process. The surge in the luteinizing hormone triggers ovulatory changes in oocyte microenvironment. In this review, we discuss how proteolytic cleavage affects formation of cumulus ECM following hormonal stimulation; in particular, how the specific proteasome inhibitor MG132 affects gonadotropin-induced cytoskeletal structure, the organization of cumulus ECM, steroidogenesis, and nuclear maturation. We found that after the inhibition of proteolytic cleavage, gonadotropin-stimulated oocyte-cumulus complexes (OCCs) were without any signs of cumulus expansion; they remained compact with preserved cytoskeletal F-actin-rich transzonal projections through the oocyte investments. Concomitantly, a significant decrease was detected in progesterone secretion and in the expression of gonadotropin-stimulated cumulus expansion-related transcripts, such as HAS2 and TNFAIP6. In agreement, the covalent binding between hyaluronan and the heavy chains of serum-derived the inter-alpha-trypsin inhibitor, essential for the organization of cumulus ECM, was missing.

Keywords: extracellular matrix; hyaluronan; oocyte–cumulus complex; proteasome.

Figure 1

Immunofluorescence analysis of IαI components in the oocyte–cumulus ECM. Oocyte-cumulus complexes (OCCs) were cultured for 20 h without (A) or with (B–E) FSH (100 ng/mL) in the absence (A,B; in PVP-supplemented medium) or in the presence of serum (5 % FBS) (C–E, magnification of ECM structure present in C). In the absence of serum (A,B), the cumulus cells remain close to each other and to the oocyte, and the ECM is absent. In contrast, in the presence of serum and FSH (C–E, magnification of the cumulus ECM structure present in C), cumulus cells synthesize and organize an ECM structure that contains components immunoreactive for IαI and present as a network of cables. The immunofluorescence analysis was performed by using a rabbit anti-human IαI antibody (dilution 1:100; DAKO, Carpenteria, CA, USA) and Alexa Fluor 488 goat anti-rabbit IgG as a secondary antibody (dilution 1:500; Molecular Probes, green). Nuclei are in red.

Figure 2

Confocal microscope analysis of IαI in the oocyte-cumulus complexes. Oocyte–cumulus cells complexes were analyzed at the beginning (0 h) and at the end (20 h) of the culture carried out in the presence of 5 % FBS, with or without FSH (100 ng/mL) stimulation, as indicated. Immunoreactivity for IαI components was evident in the ECM structure organized around the cumulus cells after FSH stimulation by using a rabbit anti-human IαI antibody (Ab dilution 1:100; DAKO, Carpenteria, CA, USA) and Alexa Fluor 488 goat anti-rabbit IgG as a secondary antibody (dilution 1:500; Molecular Probes, green). The boundary of the oocyte is marked by a dashed line.